Method of diagnosing metabolic bone diseases

ABSTRACT

A method of diagnosing metabolic bone diseases, especially osteoporosis and arthrosis characterized by determining the concentration of osteoclastgenesis inhibitory factor (OCIF) in humor. Monoclonal antibodies recognizing equally both of monomer type and dimer type of OCIF. Monoclonal antibodies recognizing selectively dimer type of OCIF. And to provide an assay kit for determination of OCIF concentration comprising the aforementioned two antibodies recognizing different epitope of OCIF and having high affinity showing dissociation constant of less than 2×10 −7  M with antigen. It is useful for a method of diagnosing metabolic bone diseases, especially osteoporosis and arthrosis or for an assay reagent for research thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/308,800, filed May 24, 1999, now U.S. Pat. No. 6,693,175 which is anational phase under 35 U.S.C. § 371 of International Application No.PCT/JP98/03421, filed Jul. 31, 1998, incorporated herein by reference,which claims priority to Japanese Application No. 276475/1997, filedSep. 24, 1997.

TECHNICAL FIELD

The present invention relates to a method of diagnosing metabolic bonediseases, especially osteoporosis and arthral diseases. In addition, thepresent invention relates to monoclonal antibodies used in the diagnosisand kits for diagnosis using the monoclonal antibodies. The presentinvention is useful as a method of diagnosing metabolic bone diseases,especially osteoporosis and arthral diseases, or as assay reagents forresearch use thereof.

BACKGROUND ART

Bone metabolism depends on integrated activity of osteoblasts that formbone and osteoclasts that resorb bone. In a healthy adult, the balanceof bone formation and bone resorption is kept and the bone mass ismaintained constant. Metabolic bone diseases are thought to develop bylosing this balance. As metabolic bone diseases, osteoporosis,hypercalcemia, Pajet's disease, renal osteodystrophy, rheumatoidarthritis and osteoarthritis etc. are known. Osteoporosis is exemplifiedas a typical metabolic bone disease. Osteoporosis is thought to be adisease accompanied with decrease in bone mass and shows clinicalsymptoms, such as bone fracture or bone pain (lumbago and/or dorsalgia)caused by decrease in bone mass. Decrease in bone mass is induced byvarious causes such as aging after growing period, bone metastasis, orhyperthyroidism. As a method of diagnosing osteoporosis, bone mineralmass and/or bone density are determined by an apparatus to measurephysical bone mass, such as X-ray diffraction (MD method), DPA (Dualphoton absorptiometry), DEXA (Dual energy X-ray absorptiometry), CXD(Computed X-ray Densitometry) and low-frequency supersonic waves. Thecriterion of osteoporosis using these diagnostic methods is alwaysrectified depending on technical revolution.

The risk of bone fracture in future might be surely predicted bydecrease in bone mineral mass and/or bone density. However, decrease inbone mineral mass and/or bone density is not a sole risk factor of bonefracture and risk of bone fracture is thought to increase by phenomenaaccompanying with aging such as decrease in elasticity of collagenfiber, qualitative deterioration of bone structure, lowered muscularstrength. At presentrisk factor except lowered muscular strength can notbe measured non-invasively and non-invasive measurement is an importantobject to be solved in future. Further, decrease in bone mineral massand/or bone density is just a result of losing the balance of bonemetabolism and neither a cause of the disease or a diagnostic parameterthereof.

As supplement covering these defects of measurement of bone density,measurement of serum level and/or urinary excretion of factorsregulating bone metabolism (parathyroid hormone (PTH), active form ofvitamin D₃ and calcitonin etc.), various kinds of factor released frombone tissue accompanying with bone remodeling (bone alkaline phosphates,acid phosphatase, pyridinoline, deoxypyridinoline, type-I procollagenpeptide, osteocalcin etc.) are tried to use to diagnose the disease.These factors would reflect of bone metabolic state at the time ofmeasurement and are expected as an early parameter of bone loss and theextent thereof. However, as for these markers of bone metabolism, thereare still problems, for example, they do not express local bonemetabolic change, they can be affected by diet or circadian rhythm, sothat changes in the level of these above factors do not necessarilyreflect specific changes in bone metabolism. From these situations,development of highly specific and precise measurement of a novel markerinvolved in bone metabolism is expected for establishment of methods ofsuitable diagnosis, prevention and treatment of various kinds ofmetabolic bone diseases such as osteoporosis.

The present inventors found that osteoclastgenesis inhibitory factor(OCIF) was present in a culture medium of human fetal lung fibroblasts,IMR-90 (ATCC CCL186) and succeeded in isolation thereof. In addition,the inventors also succeeded in cloning of cDNA encoding this proteinand confirmed the usefulness thereof as an agent for improving bonemetabolism by evaluating pharmacological effect of recombinant OCIF(rOCIF) in vitro and in vivo (WO 96/26217). Further, the presentinventors confirmed that administration of rOCIF significantly improvedbone density and bone strength in various kinds of animal model ofmetabolic bone disease and that administration of a large amount ofrOCIF also significantly increased bone mass and bone volume in a normalanimal without was not any side-effect in the examination of variousorgans other than osseous tissue, hematology and clinical biochemistryand hemolytic cell. From the results of in vivo experiment, it was foundthat OCIF is a highly tissue-specific cytokine having an action only onosseous tissue. In addition, the present inventors confirmed that, in ananimal cell, OCIF was secreted as homodimer form of OCIF with amolecular weight of about 120 kDa and the homodimer type of OCIF wasconverted into monomer form of OCIF with a molecular weight of about 60kDa by protease processing. And as it was confirmed that both types ofOCIF were present in a culture medium of human cell line (Tsuda etal.:Biochem. Biophys. Res. Commun. 234, 137–142)(1997)), it is expectedthat both types of OCIF are present in humor of mammal including humanbeing.

Accordingly, for elucidating whether or not OCIF can be a novel markerof bone metabolism, it is necessary to study precisely the correlationbetween various kinds of metabolic bone diseases and the level of eachtype of OCIF or total concentration of both types of OCIF in one ofpatients with metabolic bone diseases. Therefore, an antibodyrecognizing equally both types of OCIF and an antibody recognizing onlyhomodimer are required for the above purpose. Any anti-OCIF monoclonalantibody having such features has not been obtained yet.

DISCLOSURE OF THE INVENTION

Considering these situations, the present inventors eagerly studied andfound monoclonal antibodies with markedly high affinity (dissociationconstant thereof was less than 10⁻⁹ M) recognizing equally both ofmonomer- and homodimer-types of OCIF and monoclonal antibodiesrecognizing specifically homodimer type of OCIF. Further, the inventorsconstructed a highly sensitive enzyme immunoassay kit (sandwich ELISA)using these antibodies. As the results of measurement of serumconcentration of OCIF in young adults, the aged, patients withosteoporosis, hyperthyroidism and various kinds of disease includingcancer using the sandwich ELISA, a high inverse-correlation was foundbetween serum concentration of OCIF and bone density. As the results ofmeasurement of the concentration of OCIF in synovial fluid of patientswith arthrosis such as rheumatoid arthritis, osteoarthritis, trauma andgouty seizure etc., OCIF concentration in synovial fluid of a patientwith progressed joint destruction was found to be significantly low.

OCIF was found to be useful as a novel diagnostic marker of metabolicbone disease, because the determination of OCIF in serum and synovialfluid by the present sandwich ELISA makes it to precisely predict thedynamics of bone density and the progress of joint disruption,respectively, and thereby predict the decrease in bone mass and jointdisruption at early stage of these bone decreases. Accordingly, anobject of the present invention is to provide a method of diagnosingmetabolic bone disease, especially osteoporosis, and joint destructioncaused by rheumatism, characterized by determination of theconcentration of human osteoclastgenesis inhibitory factor andmonoclonal antibodies used therein and a kit for measurement of OCIFusing the antibodies.

The present invention relates to a method of diagnosing metabolic bonedisease by determination of the concentration of osteoclastgenesisinhibitory factor (OCIF) in sample humor.

The diagnosis of the present invention is especially useful fordiagnosis of osteoporosis and arthrosis. As humor, serum or synovialfluid can be used. Diagnosis of osteoporosis can be carried out bydetermination of serum concentration of OCIF. And diagnosis of arthrosiscan be carried out by determination of the concentration of OCIF insynovial fluid. Diagnosis by the present invention is especially usefulfor diagnosing osteoporosis. As a humor, serum or synovial fluid can beused.

In addition, the present invention relates to monoclonal antibodies usedin the diagnosis. As monoclonal antibodies, a monoclonal antibodyrecognizing equally both of monomer- and dimer-types of OCIF and anantibody recognizing selectively only dimer type of OCIF can beexemplified. Further monoclonal antibodies include high affinityantibodies recognizing a different epitope and having dissociationconstant of less than 2×10⁻⁷ M with antigen.

Further, the present invention relates to a kit for OCIF determinationcomprising these monoclonal antibodies. The diagnostic method of thepresent invention can be carried out by taking humor such as blood(serum), synovial fluid from an object of diagnosis and measuring OCIFby a kit for OCIF determination using the above monoclonal antibodies.

The monoclonal antibodies can be obtained by the method described below.That is, as an antigen for immunization being necessary for preparationof anti-OCIF monoclonal antibody, human OCIF isolated from culturemedium of human embryonic lung fibroblasts, IMR-90 according to a methoddescribed in WO96/26217 can be also used. Recombinant human OCIF can bealso used. Recombinant human OCIF can be obtained by inserting cDNA ofhuman OCIF into an expression vector in a conventional manner andexpressing it in animal cell such as CHO cell, BHK cell and Namalwa celletc., or insect cell followed by purification. According to the methodof Tsudaet al. (Biochem. Biophys. Res. Common. 234, 137–142 (1997)),monomer- and dimer-types of OCIF can be purified by reverse-phasechromatography, respectively. Further, both types of OCIF can berespectively purified by a combination of SP-Sepharose, sulfatedCellurofine and resource S column chromatography in replace ofreverse-phase chromatography. Spleen cells prepared from mammalimmunized with the antigen or lymphocytes immunized in vitro can befused with myeloma cell line to make a hybridoma. Using highly purifiedmonomer- and homodimer-types of OCIF as antigens and culture media ofthe above hybridoma, the cell line can be established by screeninghybridoma producing an antibody recognizing equally both types of OCIFor an antibody recognizing specifically only homodimer type of OCIFfollowed by cloning the hybridoma. Further, the aimed antibodies can beobtained by culturing the established and stable hybridoma.

When mammal is immunized for preparation of hybridoma, though animalspecies is not limited, small animal such as mice or rat is generallyused. In immunization, OCIF as an antigen can be diluted inphysiological saline solution to suitable concentration and the solutionthereof can be administered intravenously or intraperitoneally, ifnecessary, Freund's complete adjuvant can be administered therewith,generally, 3–4 times at 1–2 weeks intervals in animal. On preparation ofhigh affinity anti-OCIF monoclonal antibody (dissociation constantthereof is less than 2×10⁻⁷), immunization can be carried out 3 times atintervals of one week and, further, immunization with antigen togetherwith Freund's incomplete adjuvant can be carried out another 4 times atintervals of one week in order to obtain the aimed monoclonal antibodyeasily and to enhance titer of OCIF in blood as much as possible.Immunized animals described above can be anatomized 3 days after thefinal immunization and spleen can be dissected. Splenocytes can be usedas immunized cell. As myeloma cell lines derived from mice to behybridized with the immunized cell, p3/×63-Ag8, p3-U1, NS-1, MPC-11,SP-2/0, FO, p3×63 Ag8, 653 and S194 can be exemplified. Further, as acell line derived from rat, R-210 can be exemplified.

To produce human antibody, human lymphocytes can be immunized in vitroand cell-fused with human myeloma cells or a human lymphocyte cell linetransformed by EB virus. Fusion of immunized cells with myeloma cellline can be carried out according to a conventional method, for example,the method of Koehler and Milstein et al. (Koehler et al., Nature, 256,495–497, 1975) but electric pulse method can be also used. Immunizedlymphocytes and a myeloma cell line can be mixed at a usual rate in cellnumber and polyethylene glycol can be added to cell culture media usedgenerally (not including Fetal calf serum, FCS) to carry out cellfusions and fused cells (hybridoma) can be selected by culturing in HATselective medium containing FCS.

Hybridomas producing a monoclonal antibody recognizing equally both ofmonomer- and homodimer-types of OCIF and an antibody recognizingselectively homodimer type of OCIF can be selected according to a methodof detecting antibody such as ELISA, plaque assay, ouchterlony method oragglutination method. ELISAs using purified monomer- and homodimer-typesof OCIF can detect the object antibody very easily and precisely. It wasdifficult to obtain high affinity antibody (dissociation constant isless than 2×10⁻⁷M) by usual solid phase ELISA. That is, when usual solidphase is used, culture media of hybridoma (50–100 μl) is placed into 96well immunoplates coated with antigen (Nunc) to proceed the primaryreaction and, then, enzyme labeling, for example, peroxidase (POD)labeled anti-mouse IgG antibody is added to proceed the secondaryreaction. Then, the solution of enzyme substrate (50–100 μl) is added toeach well in the immunoplates to complete enzymatic reaction andabsorbance in each well is determined. Culture media of hybridomashowing high absorbance can be thought not only to produce a largeamount of low affinity antibody but also to produce a high affinityantibody even if productivity of the antibody is low. It is not possibleto determine which case is true.

Therefore, in the present invention, in order to recognize hybridomaproducing a high affinity antibody, usual solid phase ELISA was improvedas described below. That is, to each well of in 96 well immunoplatescoated with an antigen, human serum or bovine serum was added, followedby the addition of a small amount of culture media of hybridoma to eachwell to proceed the primary reaction under the presence of about 80–90%of serum. Under such conditions, hybridomas producing antibodies withlow affinity for the antigen, even if producing of the antibodies arehigh, can be excluded. Thus, the modified solid ELISA made it possibleto selectively screen by hybridomas producing antibodies with highaffinity for the antigen. Using the improved solid phase ELISA,hybridomas producing antibodies recognizing equally both of monomer- andhomodimer-types of OCIF as antigens and another hybridomas producingantibodies recognizing specifically homodimer type of OCIF can beselected and stable hybridoma producing each antibody can be establishedby cloning 3–5 times by limited dilution method. Hybridoma establishedlike this can be subcultured by culturing method usually used andpreserved by freezing, if necessary. Hybridoma can be cultured by usualmethod and antibody can be recovered from the culture media. Further,antibody can be recovered from ascites derived from mammal in whichhybridoma is implanted intraperitoneally. Antibody in culture media orin ascites can be purified by usual method of purifying antibody such assalting out, ion exchange or gel permeation chromatography, protein A orG affinity chromatography. Obtained antibody is an antibody recognizingequally both of monomer- and homodimer-types of OCIF and an antibodyrecognizing selectively homodimer type of OCIF. Each antibody can beused for measurement of the amount of OCIF (monomer type ofOCIF+homodimer type of OCIF) and the amount of homodimer type of OCIF.These antibodies can be labeled by radioactive isotope or enzyme andused in radioimmunoassay (RIA) or enzymeimmunoassay (ELISA) to determinethe amount of OCIF (the amount of monomer type of OCIF+homodimer type ofOCIF) or the amount of homodimer type of OCIF only. Especially, theantibody of the present invention recognizing selectively homodimer typeof OCIF can make it clear that there are different epitope (s) inmonomer- and homodimer-types of OCIF and recognize the epitope presentonly in homodimer type of OCIF which is absent in monomer type of OCIF.

The amount of OCIF and the amount of homodimer type of OCIF can bedetermined by using an antibody obtained by the present inventionrecognizing equally both ofmonomer- and homodimer-types of OCIF as asolid phase antibody, and by using radioactive isotope or enzyme-labeledlabeling antibody recognizing equally both of monomer- andhomodimer-types and antibody recognizing selectively only homodimer typeof OCIF with radioactive isotope or enzyme as secondary antibody,respectively.

Further, when only the amount of homodimer type of OCIF is wanted todetermine, as a solid phase antibody, OI-26 antibody recognizingselectively homodimer type of OCIF as described in example 6 (table 1)and, as labeled antibody, OI-19 or OI-4 antibody recognizing equallyboth of monomer- and homodimer-types of OCIF can be also used. By usingthese assay systems, the amount of OCIF or only that of homodimer typeof OCIF in body fluids such as blood, urine and synovial fluid etc. orin cell culture media can be determined.

A kit of the present invention comprises (i)any one of primary antibodyand secondary antibody is OI-19 or OI-26 anti body, and (ii) the otherantibody is OI-4 antibody and usual combination of reagents used inusual sandwich method. That is, an immunoassay kit comprises (1) primaryantibody immobilized on insoluble carrier, (2) labeled secondaryantibody, (3) solubilizer, (4) washing agent, and (5) substrateandreaction stopping reagent to determine enzymatic activity in the caseof enzyme labeling. As insoluble carrier, polystyrene, polyethylene,polypropylene, polyester, polyacryronitrile, fluorinated resin,crosslinked dextran, polysaccharide, latex, latex polymer containingmagnetic particles plated with metal etc., paper, glass, metal, agaroseand combination of the above carries can be exemplified. As the shape ofinsoluble carrier, tray, sphere, fiber, stick, plate, container, cell,test tube, porous filter can be used. Further, as labeling materialsused for preparation of labeled antibody, enzymes, fluorescentsubstances, luminescent substances and radioactive substances can beadvantageously used. As enzymes, peroxidase, alkaline phosphatase,β-D-galactosidase, glucose oxidase, malate dehydrogenase,glucose-6-phosphate dehydrogenase, invertase can be used. As fluorescentsubstances, fluorescein isothiocyanate and phycobili-protein can beused. As luminescent substances, isolucinol and lucigenin can be used.And, as radioactive substances, I¹²⁵, I¹³¹, C¹⁴, H³ can be exemplified.These above examples are merely examples and anything used inimmunoassay can be used.

When a labeling material is enzyme, substrate and, if necessary, colordeveloper can be used to determine enzymatic activity. When peroxidaseis used as an enzyme, H₂O₂ is used as a substrate and, as colordeveloper, 2,2′-azinodi[3-ethylbenzthiazoline sulfonic acid] ammoniumsalt(ABTS), 5-aminosalicylic acid, o-phenylenediamine,4-aminoantipyrine, 3,3′,5,5′-tetramethylbenzidine, homocevadillinicacid, and tyramine can be used.

And when alkaline phosphatase is used as an enzyme,o-nitrophenylphosphate and 4-methylumbelliferylphosphate can be used asa substrate. When β-D-galctosidase is used as an enzyme,fluoroscein-di-(β-D-galctopyranoside),4-methylumbelliferyl-β-D-galctopyranoside can be used as a substrate.

As a solubilizer disclosed in (3) in the above immunoassay kit, any oneused usually in immunoassay can be used, and for example, phosphatebuffer solution, tris-HCl buffer solution, acetic acid buffer solutionwith pH of 6.0–8.0 can be respected as appropriate examples. Further, asa washing agent disclosed in (4), any one used generally in immunoassaycan be used. For example, physiological saline solution, phosphatebuffer solution, tris-HCl buffer solution and mixed solution thereof canbe exemplified. Further, to the above washing agent, nonionic surfactantsuch as Triton X-100, Tween 20 or Brij 35 or ionic surfactant such assodium dodesyl sulfate or CHAPS can be added.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows standard curve of ELISA using OI-19 antibody and OI-4antibody in example 7.

(Explanation of Code)

∘: homodimer type of OCIF

•: monomer type of OCIF

FIG. 2 shows the calibration of ELISA using OI-26 antibody and OI-4antibody in example 7.

(Explanation of Code)

∘: homodimer type of OCIF

•: monomer type of OCIF

FIG. 3 shows the blood concentration of OCIF in patients withosteoporosis and in healthy persons in example 8.

FIG. 4 shows the correlation between the urine concentration ofpyridinoline and the blood concentration of OCIF in example 8.

FIG. 5 shows the correlation between the urine concentration ofdeoxypyridinoline and the blood concentration of OCIF in example 8.

FIG. 6 shows the concentration of OCIF in synovial fluid of patientswith arthrocele in example 9.

(Explanation of Code)

RA: rheumatoid arthritis

OA: osteoarthritis

Tr: trauma

G: gouty seizure

BEST MODE FOR PRACTICE OF THE INVENTION

The present invention will be described in more detail by showingexamples. However, these are merely examples and the scope of thepresent invention will not be limited by these examples.

EXAMPLE 1

Purification of Monomer Type of OCIF or Homodimer Type of OCIF as anAntigen

OCIF-producing CHO cells described in WO96/26217 were seeded in EX-CELL301 medium (JRH Bioscience) at a cell density of 1×10⁵ cells/ml andcultured at 37° C. for 7 days using a jar for cell culture (2 literscontainer). To the obtained culture medium, CHAPS(3-[(3-cholamidopropyl)-dimethylammonio-]-1-propanesul fonate, Sigma)was added to a concentration of 0.1%. After pH of the medium wasadjusted to 6.0 by acetic acid, the medium was filtered through 0.22 μmfilter (milidisk, Millipore). The culture medium was loaded on a SPsepharose HP column (2.6×10 cm, Pharmacia) equilibrated with 50 mMbis-tris-HCl buffer solution containing 0.1% CHAPS. After washing thecolumn with the same buffer solution, the column was developed with alinear gradient from 0 to 1 M NaCl for 100 minutes at a flow rate of 4ml/min. and the elute was fractionated into 8 ml each. According to themethod described in WO96/26217, OCIF activity in each fraction wasdetermined, so that OCIF fraction was obtained. After the OCIF fractionwas diluted 10-fold with 50 mM bis-tris-HCl buffer solution, pH 6.0containing 0.1% CHAPS, it was loaded on a sulfate-cellurofine column(2.6×10 cm, Seikagaku-kogyo) equilibrated with 50 mM bis-tris-HClbuffer, pH 6.0. The column was washed with 50 mM bis-tris-HCl buffersolution at pH 6.0 containing 0.1% CHAPS, followed by developing withlinear gradient from 0 to 1.5 M NaCl for 100 minutes at flow rate 4 ml/min. and fractionating into 8 ml each. OCIF activity in each fractionwas determined as described above.

A portion of each fraction was subjected to SDS-PAGE under non-reducingconditions and fractions having OCIF activity and molecular weight of 60kDa were collected and the OCIF pool was named fraction 1. In addition,fractions having OCIF activity and showing molecular weight of 120 kDaunder non-reducing conditions were collected and the OCIF pool was namedfraction 2. Fraction 1 and fraction 2 were diluted 10-foled with 50 mMtris-HCl buffer, pH 7.0, containing 0.1% CHAPS, respectively and eachfraction was loaded on a RESOURCE S column (0.64×3 cm, Pharmacia)equilibrated with 50 mM tris-HCl buffer, pH7.0, containing 0.1% CHAPS.After washing the column with 10 mM sodium phosphate buffer, pH 7.0containing 0.01% polysorbate, the column was developed with a lineargradient from 0 to 0.6 M NaCl for 15 minutes at flow rate of 1 ml/min.and fractionating into 0.5 ml each. OCIF activity in each fraction offraction 1 and fraction 2 was determined as described above andfractions having OCIF activity were collected, so that monomer type ofOCIF from fraction 1 and homodimer type of OCIF from fraction 2 wereobtained.

EXAMPLE 2

Immunization of Mice and Preparation of Hybridoma

Monomer- and homodimer-types of OCIF purified as described in example 1were dissolved in physiological saline solution to concentration of 100μg/ml, respectively. To a mixture each containing equal amount of bothtypes of OCIF prepared as above, the same volume of Freund's completeadjuvant was added. The mixed solution was emulsified well andadministered intraperitoneally (200 μl/mouse) to Balb/c mice 3 times atintervals of one week to immunize mice. Then, to a mixture containing 25μg/ml of each both types of OCIF, the same volume of Freund's incompleteadjuvant was added to make an emulsion, 200 μl of which was administeredper mouse to the above Balb/c mice 4 times at intervals of one week.

One week after the 4th booster immunization, 100 μl of a mixed solutioncontaining 100 μg/ml of each both types of OCIF was intravenouslyadministered to Balb/c mice. Three days after the final immunization,spleen was dissected and spleen cells were isolated and fused with mousemyeloma cells, P3×63-AG8.653 (ATCC CRL-1580), according to theconventional method (Koehler,G. and Milstein, C., Nature, 256.495(1975)). After cell fusion, cell suspension was cultured in HAT mediumcontaining hypoxanthine, aminopterin and thymidine for 10 days. Afterhybridoma appeared in replace of myeloma cells, cell culture was kept onculturing by changing culture medium into HT medium excludingaminopterin from HAT medium.

EXAMPLE 3

Selection and Cloning of Hybridoma

Ten days after the cell fusion, hybridomas appeared. Hybridoma producinghigh affinity antibody recognizing equally both of monomer- andhomodimer-types of OCIF and hybridomas producing antibody recognizingselectively homodimer type of OCIF were screened. That is, monomer- andhomodimer-types of OCIF were dissolved in 0.1 M sodium bicarbonate (pH9.6) to a concentration of 5 μg/ml, respectively and 50 μl of eachantigen solution was added to each well in 96 well immunoplates (Nunc)and the plates were kept at 4° C. overnight to coat each well with eachantigen. Antigen solution in each well was discarded and each well waswashed with phosphate buffered saline containing 0.1% polysorbate 20(PBS-P) and 40 μl of bovine fetal serum was added to each well.

Then, 10 μl of conditioned medium of hybridoma was added to each welland then the plates were incubated under 80% serum concentration at roomtemperature for 2 hours. After the incubation, plates were washed withPBS-P and 50 μl of peroxidase labeled anti-mouse IgG (KPL) diluted5000-fold with physiological saline solution containing 25% BLOCKAGE wasadded to each well and the plates were incubated at room temperature for2 hours. After washing the plates with PBS-P, 50 μl of enzyme substratesolution (tetramethylbenzidine (TMB), ScyTek) was added to each well forcolor developing. Subsequently the enzymatic reaction was terminated byadding 50 μl of stopping reagent, (ScyTek). Hybridomas producingantibodies were screened by determing absorbance at 450 nm of each wellusing microplate reader (an immunoreader NJ2000, Nihon-intermed).Hybridomas showing high absorbance and producing antibody recognizingequally both of monomer- and homodimer-types of OCIF, and hybridomasshowing high absorbance and producing antibody recognizing selectivelyhomodimer type of OCIF were screened, respectively. Stable hybridomaclone producing hybridoma cell line was established by repeating cloning3–5 times from each hybridoma by limiting dilution method. Amonghybridomas producing antibodies, hybridomas with high productivity ofthe object antibodies were selected.

Thus, the hybridomas, OI-19 and OI-4 which produced the antibodies,OI-19 and OI-4, respectively, recognizing equally both of monomer- andhomodimer-types of OCIF were obtained. In addition, the hybridoma, OI-26which produced OI-26 antibody, OI-26, recognizing selectively homodimertype of OCIF was obtained. These hybridomas were deposited at NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology and the deposit number for OI-4, OI-19 and OI-26was FFERM BP-6419, FERM BP-6420 and FERM BP-6421, respectively.

EXAMPLE 4

Production and Purification of Monoclonal Antibody

Hybridoma producing high affinity antibody recognizing equally both ofmonomer- and homodimer-types of OCIF and hybridoma producing antibodyrecognizing selectively homodimer type of OCIF obtained in example 3were cultured, respectively, and 1×10⁶ cells of hybridoma per mouse wereintraperitoneally administered to Balb/c mice in which pristine(Aldrich) had been administered. Two weeks after administration,accumulated ascites containing monoclonal antibody of the presentinvention was collected. Purified antibody was obtained by protein Acolumn (Pharmacia) chromatography.

EXAMPLE 5

Determination of Dissociation Constant (Kd Value) of Monoclonal Antibody

According to the method of Betrand Friguet (Journal of ImmunologicalMethods, 77, 305–319, 1986), dissociation constant of monoclonalantibody was determined. That is, purified antibody obtained in example4 was diluted in 0.2 M tris-HCl pH 7.4 (first buffer) containing 40%BlockAce (Snow Brand Milk Products) and 0.1% polysorbate 20 to aconcentration of 5 ng/ml. To the above solution, the same volume of 6.25ng/ml–10 μg/ml of purified monomer type of recombinant OCIF or homodimertype of recombinant OCIF which was obtained in example 1, diluted withthe first buffer was mixed and the mixture was kept it at 4° C. for 15hours, so that OCIF bound to monoclonal antibody. At 15 hours afterthen, dissociation constant of monoclonal antibody against monomer typeof OCIF or homodimer type of OCIF was determined by measuring antibodyunbound to OCIF using solid phase ELISA in which monomer type of rOCIFor homodimer type of rOCIF (10 μg/ml, 100 μl/well) was immobilized.

EXAMPLE 6

Assay of Class and Subclass of Monoclonal Antibody

The class and the subclass of monoclonal antibody of the presentinvention was assayed using immunoglobulin class and subclass assay kit(Amersham). The assay was carried out according to the protocol directedin a kit. The results obtained in example 5 and 6 were shown in table 1.

TABLE 1 Dissociation dissociation constant constant Antibody subclass(for monomer) (for dimer) OI-4  IgG₁ (κ) 7.3 × 10⁻¹⁰ 9.9 × 10⁻¹² OI-19IgG₁ (κ) 7.0 × 10⁻¹⁰ 1.2 × 10⁻¹¹ OI-26 IgG₁ (κ) — 1.5 × 10⁻⁷ 

From these results, the antibodies, OI-4 and OI-19, were found to beantibody recognizing equally both of monomer- and homodimer-types ofOCIF and antibody OI-26 was found to be antibody recognizing selectivelyonly homodimer type of OCIF. Further, all antibodies belonged to IgG1and were found to be antibodies with extremely high affinity havingdissociation constant of less than 2×10⁻⁷ M for monomer- andhomodimer-types of OCIF.

EXAMPLE 7

Determination of OCIF by ELISA

Sandwich ELISA was constructed by using 3 kinds of antibody obtained asdescribed above, that is, antibody OI-4, OI-26 and OI-19 as immobilizedantibody and labeled antibody. Labeling antibody was carried out usingmaleimide activated peroxidase kit(Pias). The Antibody, OI-19 as aprimary antibody in the case of ELISA measuring both of monomer- andhomodimer-types of OCIF or the antibody, OI-26 as a primary antibody inthe case of ELISA measuring selectively homodimer type of OCIF wasdissolved in 0.1 M sodium bicarbonate (pH 9.6) to a concentration of 10μg/ml, respectively, 100 μl of which was added to each well in 96 wellimmunoplates (Nunc) and the plates were kept at 4° C. overnight toimmobilize antibody onto each well. The solution in each well wasdiscarded and 300 μl of 50% BlockAce (Snow Brand Milk Products) wasadded to each well to block at room temperature for 2 hours. Afterblocking, plates were washed with phosphate buffered saline (PBS-P)containing 0.1% polysorbate 20. Monomer- and homodimer-types of OCIFwere dissolved in 0.2 M tris-HCl (pH 7.4) containing 40% BlockAce (SnowBrand Milk Products) and 0.1% polysorbate 20 (primary buffer),respectively, and then diluted to prepare each type of OCIF solutionswith various concentrations.

To each well, 100 μl of the solutions of monomer type or homodimer typeof OCIF with various concentrations was added to react at roomtemperature for 2 hours. Two hours after, plates were washed with PBS-Pand POD-labeled OI-4 antibody diluted in 0.1 M Tris-HCl (pH 7.4)containing 25% BLOCKACE and 0.1% polysorbate was added to each well inthe plates as an antibody recognizing equally both of monomer- andhomodimer-types of OCIF and the plates were allowed to stand at roomtemperature for 2 hours. Plates were washed with PBS-P and 100 μl ofenzyme substrate solution (tetramethylbenzidine (TMB), ScyTek) was addedto each well. After developing, 100 μl of stopping reagent, (ScyTek) wasadded to each well to stop enzymatic reaction. Absorbance at 450 nm ofeach well was determined using a microplate reader. The results in thecase of using the antibody, OI-19, recognizing equally both of monomer-and homodimer-types of OCIF as a primary antibody was shown in FIG. 1and the results in the case of using the antibody, OI-26, recognizingselectively homodimer type of OCIF as a primary antibody was shown inFIG. 2.

As a result, as shown in FIG. 1, detection sensitivity of sandwich ELISAwas about 25 pg/ml and found to be able to determine extremely smallamount of OCIF when the antibody, OI-19 recognizing equally both ofmonomer- and homodimer-types of OCIF was used as an immobilized antibodyand the antibody, OI-4 recognizing equally both types of OCIF was usedas a POD-labeled antibody. Further, as shown in FIG. 2, detectionsensitivity of sandwich ELISA was 50 pg /ml and found to be able todetect selectively homodimer type of OCIF with high sensitivity when theantibody, OI-26, recognizing selectively homodimer type of OCIF was usedas an immobilized antibody and the antibody, OI-4, recognizing equallyboth of monomer- and homodimer-types of OCIF was used as a POD-labeledantibody.

EXAMPLE 8

Determination of Serum OCIF in Healthy Persons and in Patients withOsteoporosis

Serum concentration of OCIF in healthy persons and in patients withosteoporosis (based on the criterion made by Japanese Society for Boneand Mineral Research) was determined using a partly improved ELISAsystem of OCIF (monomer type of OCIF+homodimer type of OCIF) describedin example 7. That is, for determination of OCIF (monomer type ofOCIF+homodimer type of OCIF), the antibody, OI-19, recognizing equallyboth types of OCIF was immobilized onto 96 well immunoplate as describedin example 7 and 50 μl of the first buffer (0.2 M tris-HCl, pH 7.4containing 40% BlockAce and 0.1% polysorbate 20) containing 20 μg/mlpurified mouse IgG was added to each well. Subsequently, 50 μl of humanserum diluted 4-fold with the first buffer was added to each well andthe plates were allowed to stand at room temperature for 2 hours. Afterthe plates were washed with PBS-P 6 times, 100 μl of the solution ofPOD-labeled antibody, OI-4, recognizing equally both types of OCIFdiluted 3000 times with secondary buffer (0.1 M tris-HCl, pH 7.4containing 25% BlockAce and 0.1% polysorbate 20) containing 10 μg/mlpurified mouse IgG was added to each well and the plates were allowed tostand at room temperature for 2 hours. The plates were washed 6 timeswith PBS-P and 100 μl of enzyme substrate solution (TMB, SciTech) wasadded to each well. The plates were allowed to stand at room temperaturefor 20 minutes to proceed enzymatic reaction and the reaction wasstopped by adding 100 μl of stopping reagent (ScyTek) to each well.Absorbance at 450 nm of each well was determined using a microplatereader. As for the first buffer containing the known amount of OCIF, thesame procedure as the above was taken and standard curve of OCIF wasmade as in FIG. 1. Serum concentration of OCIF was determined fromabsorbance of serum sample.

The results of determination of serum concentration of OCIF (monomertype of OCIF +homodimer type of OCIF) in healthy persons and that inpatients with osteoporosis were shown in FIG. 3. Statistical analysis ofsignificant difference of the results in FIGS. 3–5 was carried out byStudent's non-paired t test. As the results, there was a significantdifference between serum concentration of OCIF (monomer type ofOCIF+homodimer type of OCIF) in healthy persons and that in patientswith osteoporosis. Serum concentration of OCIF in patients withosteoporosis was higher than that in healthy persons. Accordingly, OCIFwas found to be a novel marker of diagnosing osteoporosis becausepathogenesis of osteoporosis could be followed by determination of serumconcentration of OCIF.

In addition, relationships between serum OCIF (monomer type ofOCIF+homodimer type of OCIF) and urine pyridinoline concentrations, andbetween serum OCIF and urine deoxypyridinoline concentrations in healthyvolunteers and patients with osteoporosis are shown in FIGS. 4 and 5,respectively. Pyridinoline concentration of 42 pmol/μmol Cr (the amountof pyridinoline pmol per creatinine 1 mol) and deoxipyridinolineconcentration of 6.2 pmol/μmol Cr are the upper limit of normal level inJapanese.

From the results, serum concentration of OCIF in patients showing higherconcentration of pyridinoline and/or deoxypyridinoline was significantlyhigher. Pyridinoline and deoxypyridinodine are cross-linked molecules ofcollagen, produced in bone substrate after incorporation of collageninto bone substrate, and released by bone destruction which is caused bybone resorption. These molecules are thought, to be a highly specificmarker of bone resorption and used widely for evaluation of clinicalobject. Since correlation between OCIF concentration and the two markerwas recognized, serum concentration of OCIF was found to be useful as amarker of bone metabolism.

EXAMPLE 9

Determination of OCIF Concentration in Synovial Fluid of Patients withArthrocele

Synovial fluid samples were taken from patients with rheumatoidarthritis (RA, 43 cases), osteoarthritis (OA, 6 cases), Trauma (Tr, 3cases) and gouty seizure (G, 6 cases) who visited hospital for treatmentof arthrosis, were diagnosed as clear arthrocele and agreed withinformed consent. OCIF concentration in the synovial fluid was measuredby a partly improved method of ELISA system of OCIF (monomer type ofOCIF+homodimer type of OCIF) described in example 8. That is, aftersynovial fluid was diluted 16-fold with the first buffer, 50 μl of whichwas added to each well in 96 well immunoplates immobilized antibodyOI-19. Other procedures than the above were the same as those in example8.

Examination results of OCIF (monomer type of OCIF+homodimer type ofOCIF) concentration in synovial fluid of patients with arthrocele wereshown in FIG. 6. For statistical analysis of data shown in FIG. 6,Kruskal-Wallis Test and Mann-Whitney Test were carried out. From theresults, OCIF concentration in synovial fluid of patient with rheumatoidarthritis was significantly lower than that of patient with goutyseizure (p=0.0023). Further, while the lowest value of OCIFconcentration in synovial fluid of patients with osteoarthritis (6cases) was 4.79 ng/ml, the number of patients with rheumatoid arthritisshowing less than 4.0 ng/ml of OCIF concentration in synovial fluid was15 cases among 43 cases.

From these results, it was suggested that deficiency of OCIF could notsuppress formation and activity of osteoclasts in patients withrheumatoid arthritis showing lower OCIF concentration in synovial fluid.OCIF-ELISA was found to be useful for diagnosis of pathogenesisrheumatoid arthritis (RA), osteoarthritis (OA), Trauma (Tr), goutyseizure (G).

EXAMPLE 10

Correlation between OCIF Concentration in Knee Joint Humor of Patientwith Rheumatoid Arthritis(RA) and Pathogenesis

In order to study the correlation between OCIF concentration in synovialfluid and progress of joint destruction, retrospective cohort study wascarried out in 2 patients with rheumatoid arthritis.

Case 1, 66 Years Old, Male

He had subjective symptom of multiarticular pain at his 50 years old in1982. In 1983, he visited a hospital and was diagnosed as RA based onclassification criterion of RA of American College of Rheumatology.After that, since it remitted by administration of anti-rheumatoid agent(Mercaptase), administration the an anti-rheumatoid agent was stopped.In 1990, because RA recurred, administration of the anti-rheumatoidagent was restarted. On Mar. 18, 1992, his right knee joint swelled andsynovial fluid was taken by puncture. The OCIF concentration in synovialfluid was 23.6 ng/ml, which was high level (The median value of OCIFconcentration of 43 RA patients was 6 ng/ml). Plasma CRP at that timewas 5.4 mg/dl, which clearly suggested that he had also inflammation.CRP did not become negative and kept around 3 mg/dl until 1997. Knee X-pwas observed from 1983 to 1997 and main symptom was osteoarthritis. Boneerosion was not observed in digit joint of hand and hand joint.

Case 2, 60 Years Old, Female

She had subjective symptom of multiaricular pain in 1987 (49 years old).On Aug. 17, 1993, she visited a hospital and was diagnosed as RA basedon classification criterion of RA of American College of Rheumatology.At that time, her right knee joint swelled and synovial fluid was takenby puncture. OCIF concentration in synovial fluid was 3.0 ng/ml, whichwas low level and plasma CRP was 13.7 mg/dl, which showed that she hadinflammation. Since then, CRP gradually decreased by administration ofanti-rheumatoid agent (Methotrexate) to a level of 4.1 mg/dl on Mar. 18,1994 and to a level of 1.1 mg/dl on 27th June in the same year. However,according to Laresen classification (Laresen A. et al., Acta Radiol.Diag. 18, 481–491, 1977), knee X-p become worse and worse, that is,grade III on 1 Sep. in 1993, grade IV on 18 Mar. in 1994 and grade V on27th June in the same year, which showed that joint destructionprogressed clearly. And operation of replacing artificial joint on 4Oct. in 1994 was carried out.

From the results of determination of OCIF concentration in synovialfluid of the above 2 cases, that is, one with mild progress of jointdestruction and another with advanced joint destruction, determinationof OCIF concentration in synovial fluid was found to be useful forevaluation of risk rate of joint destruction by rheumatism andevaluation of therapeutic effect on joint destruction.

EXAMPLE 11

Assay Kit for Determination of Monomer- and Homodimer-Types of OCIF (80Samples)

-   1) 96 well plate immobilized the antibody, OI-19 and subsequently    blocked with BlockAce according to the method described in example    7: one plate-   2) OI-4 antibody labeled with POD according to the method in example    7:10 μl (1000-fold dilution)-   3) standard recombinant OCIF (monomer type):0.5 ng/ml 400 μl-   4) solution for dilution of sample (0.01% Tween 20 and 0.2 M    tris-HCl buffer containing 40% BlockAce, pH 7.4): 10 ml-   5) solution for dilution of labeled antibody (0.1 M tris-HCl buffer    containing 0.01% Tween 20 and 25% BlockAce, pH 7.4): 10 ml-   6) solution for washing 96 well plate (PBS(−) containing 0.1% Tween    20): 1 liter-   7) substrate solution for measurement of labeled enzyme activity    (TMB solution) and reaction stopping reagent (TMB stop reagent): 10    ml, each    Assay Kit for Determination of Dimer Type of OCIF (80 Samples)-   1) 96 well plate immobilized the antibody, OI-26 and subsequently    blocked with BlockAce according to the method described in example    7: one plate-   2) OI-4 antibody labeled with POD according to the method in example    7:10 μl (1000-fold dilution)-   3) standard recombinant OCIF (dimer type):0.5 ng/ml 400 μl-   4) solution for dilution of sample (0.01% Tween 20 and 0.2 M    tris-HCl buffer containing 40% BlockAce, pH 7.4): 10 ml-   5) diluted solution of labeled antibody (0.1M tris-HCl buffer    solution containing 0.01% Tween 20 and 25% BlockAce pH 7.4): 10 ml-   6) solution for washing 96 well plate (PBS(−) containing 0.1% Tween    20): 1 liter-   7) substrate solution for measurement of labeled enzyme activity    (TMB solution). and reaction stopping reagent (TMB stop reagent): 10    ml, each    Assay Method (Kit 1 and Kit 2)

To each well in the plate 1), 100 μl of sample diluted with the solution4) and 100 μl of serially diluted standard recombinant human OCIF 3)with the solution 4) are added, respectively. After it is kept at roomtemperature for 2 hours, each well is washed 5–6 times with 300 μl ofthe solution 6). For this washing procedure, an automatic plate washercan be used. To each well in the plate after washing, 100 μl ofPOD-labeled OI-4 antibody 2) diluted 1000-fold with the solution 5) isadded, and the plate is kept at room temperature for 2 hours. Each wellin the plate is washed 5–6 times with the solution 6). For this washingprocedure, an automatic plate washer can be used. To each well, 100 μlof enzyme substrate solution 7) is added, and the plate is kept at roomtemperature for 20–30 minutes. Subsequently, the enzymatic reaction isstopped by the addition of 100 μl of reaction stopping reagent 7).

Absorbance at 450 nm of each well is determined by a microplate reader.Using absorbance at 450 nm in the case of adding standard recombinanthuman OCIF 3) diluted serially to each well, standard curve ofrecombinant human OCIF is made. OCIF concentration of each sample can bedetermined using this standard curve.

INDUSTRIAL UTILITY

By determining the concentration of human osteoclastgenesis inhibitoryfactor in humor (blood, synovial fluid, etc.) according to the method ofthe present invention, diagnosis of metabolic bone diseases, especiallyosteoporosis and arthrosis can be carried out easily and precisely. Fordiagnosis of the present invention, because the aforementionedmonoclonal antibodies and assay kits for OCIF using the monoclonalantibodies are used, diagnosis of metabolic bone diseases, especiallyosteoporosis and arthrosis can be carried out easily and precisely asmentioned above. The present invention is useful for a method ofdiagnosing metabolic bone diseases, especially osteoporosis andarthrosis or for an assay reagent for research thereof.

REFERENCE TO MICROORGANISM

-   Name and address of depository authority to which said organisms of    the present invention were deposited    -   Name: National Institute of Bioscience and Human-Technology        -   Agency of Industrial Science and Technology        -   Ministry of International Trade and Industry    -   Address:1-3, Higasi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan    -   Date of deposition thereof to the depository authority:        -   16 Oct., 1997    -   Depository Number Given by the Depository Author: FERM BP-6419        Name and address of depository authority to which said organisms        of the present invention were deposited    -   Name: National Institute of Bioscience and Human-Technology        -   Agency of Industrial Science and Technology        -   Ministry of International Trade and Industry    -   Address:1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan    -   Date of deposition thereof to the depository authority:        -   Oct. 16, 1997    -   Depository Number Given by the Depository Author: FERM BP-6420        Name and address of depository authority to which said organisms        of the present invention were deposited    -   Name: National Institute of Bioscience and Human-Technology        -   Agency of Industrial Science and Technology        -   Ministry of International Trade and Industry    -   Address:1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan    -   Date of deposition thereof to the depository authority:        -   16 Oct., 1997    -   Depository Number Given by the Depository Author: FERM BP-6421

1. A method of detecting a highly specific marker for bone resorptionassociated with osteoporosis in a mammal, the method comprising:measuring the concentration of non-administered osteoclastogenesisinhibitory factor protein in a test blood sample isolated from saidmammal, comparing the concentration of non-administeredosteoclastogenesis inhibitory factor protein in said test blood samplewith a concentration of osteoclastogenesis inhibitory factor protein ina normal subject blood sample, wherein a higher concentration of saidnon-administered osteoclastogenesis inhibitory factor protein in saidtest blood sample relative to said normal subject blood sample, isindicative of bone resorption associated with osteoporosis.
 2. Themethod of claim 1, wherein said test blood sample is a test serum sampleand said normal subject blood sample is a normal subject serum sample.3. The method of claim 1, wherein the step of measuring theconcentration of non-administered osteoclastogenesis inhibitor factorprotein in a test blood sample further comprises: contacting the testblood sample isolated from said mammal with at least one monoclonalantibody that binds specifically to an osteoclastogenesis inhibitoryfactor protein to produce an antibody-osteoclastogenesis inhibitoryfactor protein complex; and determining the concentration of saidantibody-osteoclastogenesis inhibitory factor protein complex in saidtest sample.
 4. The method of claim 3, wherein measuring theconcentration of non-administered osteoclastogenesis inhibitor factorprotein in a test blood sample further comprises: comparing theconcentration of said antibody-osteoclastogenesis inhibitory factorprotein complex in said test blood sample to a standard protein curve todetermine the concentration of osteoclastogenesis inhibitory factorprotein.
 5. The method of claim 1, wherein said concentration ofosteoclastogenesis inhibitory factor protein in a normal subject bloodsample is a concentration of osteoclastogenesis inhibitory factorprotein referenced for normal subjects.
 6. The method of claim 1,wherein said normal subject blood sample is derived from a subject witha normal pyridinoline concentration.
 7. The method of claim 1, whereinsaid normal subject blood sample is derived from a subject with a normaldeoxypyridinoline concentration.
 8. The method of claim 6, wherein saidnormal pyridinoline concentration is less than 42 pmol/μmol creatine inurine.
 9. The method of claim 7, wherein said normal deoxypyridinolineconcentration is less than 6.2 pmol/μmol creatine in urine.
 10. A methodof detecting a highly specific marker for bone resorption associatedwith osteoporosis in a mammal in need thereof, the method comprising:measuring the concentration of non-administered osteoclastogenesisinhibitory factor protein in a test blood sample isolated from saidmammal; comparing the concentration of non-administeredosteoclastogenesis inhibitory factor protein in said test blood samplewith a concentration of osteoclastogenesis inhibitory factor protein ina normal subject blood sample, wherein a higher concentration of saidnon-administered osteoclastogenesis inhibitory factor protein in saidtest blood sample relative to said normal subject blood sample, isindicative of bone resorption associated with osteoporosis.
 11. A methodof detecting a highly specific marker for bone resorption associatedwith osteoporosis in a mammal, the method comprising: measuring theconcentration of osteoclastogenesis inhibitory factor protein in a testblood sample isolated from said mammal; comparing the concentration ofosteoclastogenesis inhibitory factor protein in said test blood samplewith a concentration of osteoclastogenesis inhibitory factor protein ina normal subject blood sample, wherein a higher concentration of saidosteoclastogenesis inhibitory factor protein in said test blood samplerelative to said normal subject blood sample, is indicative of boneresorption associated with osteoporosis in said mammal.
 12. A method ofdetecting in a mammal in need thereof a highly specific marker for boneresorption, the method comprising: measuring the concentration ofnon-administered osteoclastogenesis inhibitory factor protein in a testblood sample isolated from said mammal; comparing the concentration ofnon-administered osteoclastogenesis inhibitory factor protein in saidtest blood sample with a concentration of osteoclastogenesis inhibitoryfactor protein in a normal subject blood sample, wherein a higherconcentration of non-administered osteoclastogenesis inhibitory factorprotein in said test blood sample relative to a normal subject bloodsample, is indicative of said bone resorption in said mammal.
 13. Amethod for prognosis of joint destruction in a rheumatoid arthritispatient comprising: measuring the concentration of an osteoclastogenesisinhibitory factor protein in a test synovial fluid sample obtained fromsaid patient; and determining whether said osteoclastogenesis inhibitoryfactor protein concentration is lower than a median concentration ofosteoclastogenesis inhibitory factor protein found in synovial fluidsamples obtained from rheumatoid arthritis patients.
 14. The method ofclaim 13, wherein said determining further comprises the step ofevaluating risk rate of joint destruction by rheumatism.
 15. The methodof claim 13, wherein said patient is under going therapeutic treatmenton joint destruction.